Sitafloxacin reduces tumor necrosis factor alpha (TNFα) converting enzyme (TACE) phosphorylation and activity to inhibit TNFα release from lipopolysaccharide-stimulated THP-1 cells

Sepsis is a systemic reaction to an infection and resulting in excessive production of inflammatory cytokines and chemokines. It sometimes results in septic shock. The present study aimed to identify quinolone antibiotics that can reduce tumor necrosis factor alpha (TNFα) production and to elucidate mechanisms underlying inhibition of TNFα production. We identified quinolone antibiotics reduced TNFα production in lipopolysaccharide (LPS)-stimulated THP-1 cells. Sitafloxacin (STFX) is a broad-spectrum antibiotic of the quinolone class. STFX effectively suppressed TNFα production in LPS-stimulated THP-1 cells in a dose-dependent manner and increased extracellular signal-regulated kinase (ERK) phosphorylation. The percentage of intracellular TNFα increased in LPS-stimulated cells with STFX compared with that in LPS-stimulated cells. TNFα converting enzyme (TACE) released TNFα from the cells, and STFX suppressed TACE phosphorylation and activity. To conclude, one of the mechanisms underlying inhibition of TNFα production in LPS-stimulated THP-1 cells treated with STFX is the inhibition of TNFα release from cells via the suppression of TACE phosphorylation and activity. STFX may kill bacteria and suppress inflammation. Therefore, it can be effective for sepsis treatment.

Sitafloxacin reduces tumor necrosis factor alpha (TNFα) converting enzyme (TACE) phosphorylation and activity to inhibit TNFα release from lipopolysaccharide-stimulated THP-1 cells Tumor necrosis factor alfa Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection 1 . Sepsis was first defined as sepsis-1 in 1991 and was redefined as sepsis-3 in 2016. Sepsis was further defined as a systemic inflammatory response syndrome caused by infection 2 . Particularly during gram-negative bacterial infection, lipopolysaccharide (LPS) stimulates cells to produce inflammatory cytokines and chemokines, which can sometimes result in septic shock. Inflammatory cytokines lower the blood pressure via blood vessels dilation and blood clotting within the capillaries of organs. These effects can aid the immune system in fighting infection, but can also be harmful. Thus, drugs that are not only effective against bacterial infections but also reduce inflammatory cytokines are required to avoid such harmful effects. Treatment with such drugs may help prevent septic shock and reduce mortality. Some antibiotics such as tetracycline 3,4 , macrolide [5][6][7] and oxazolidinone 8,9 have effectively reduced the production of inflammatory cytokines.
Quinolones such as garenoxacin or moxifloxacin have also been reported to reduce inflammatory cytokines 10,11 .
Sitafloxacin (STFX) is a broad-spectrum antimicrobial agent 12 . STFX is effective against pneumococcal infections, and incidence of drug-resistant mutants is low in vitro conditions 13 . STFX was effective against Haemophilus influenzae pneumonia in a murine model 14 .
In a clinical study, STFX was also proven effective and safe in elderly patients with pneumonia including aspiration pneumonia in nursing homes 15 . STFX treatment was effective in patients with both acute complicated urinary tract infection and pyelonephritis caused by Escherichia coli producing extended-spectrum betalactamase (ESBL) 16 . Another study also reported that STFX was effective against the E. coli producing ESBL following 3 days of carbapenem therapy 17 .
STFX, a broad-spectrum oral fluoroquinolone, has been approved in Japan for the treatment of respiratory and urinary tract infections. However, whether STFX can be used for treating patients with sepsis or whether it suppresses the production of inflammatory cytokines and chemokines is unknown, which we aimed to determine in the present study.
STFX inhibited the production of chemokines. STFX inhibited not only TNFα production but also chemokines production, as indicated by additional experiments with LPS-stimulated THP-1 cells. The concentration of interleukin-8 (IL-8) in the supernatants of cells treated with 50 µg/mL STFX was significantly decreased to 10,472.00 ± 474.67 pg/mL compared with that of LPS alone (17,802.33 ± 190.07 pg/mL) (p < 0.01) (Fig. 4a). The concentrations of interferon inducible protein (IP-10) in the supernatants of cells treated with 50 µg/mL STFX was significantly decreased to 77.83 ± 9.70 pg/mL compared with that of the cells treated with LPS alone (3649.00 ± 377.59 pg/mL) (p < 0.01) (Fig. 4b). The concentration of monocyte chemoattractant protein-1 (MCP-1) in cell supernatants in the presence of 50 µg/mL STFX was also significantly decreased to 161.67 ± 11.59 pg/ mL compared with that of LPS alone (3453.00 ± 148.55 pg/mL) (p < 0.01) (Fig. 4c). Furthermore, macrophage inflammatory protein-1α (MIP-1α concentrations in the supernatants of cells followed by treatment with 50 µg/mL STFX were significantly decreased to 9336.67 ± 206.50 pg/mL compared with that of the cells treated The phosphorylated form of extracellular signal-regulated kinase (ERK) increased treated with STFX. THP-1 cells (2 × 10 5 /mL) were stimulated with LPS (0.1 µg/mL) with or without the presence of STFX (50 µg/mL) for 30 min and 60 min. The phosphorylated form of ERK increased after treatment with STFX and LPS compared with treatment with LPS alone. The phosphorylated forms of nuclear factor kappa B (NF-κB) and p38 did not decrease in the cells treated with STFX and LPS compared with those treated with LPS alone (Fig. 5). Supplementary Fig. S1 presents the full-length blot and image (online).

STFX inhibited TNFα release from cells.
THP-1 cells (2 × 10 5 /mL) were stimulated by LPS (0.1 µg/mL) with or without STFX (50 µg/mL). After 4 h of incubation, intracellular TNFα was stained with anti-TNFα antibody PE. The percentage of intracellular TNFα in the cells treated with STFX and LPS increased from 4.4 to 16.2% compared with that of the cells treated with LPS alone (Fig. 6).

Discussion
TNFα plays an important role in sepsis. TNFα blocking protected mice from sepsis symptoms 18 . Some clinical studies investigating the monoclonal antibodies produced against TNFα in patients with sepsis or septic shock have been reported [19][20][21] . The modulation of TNFα and other inflammatory cytokines and chemokines is considered important in the treatment of severe infectious diseases, especially sepsis or septic shock. Some types of antibiotics can modulate inflammatory cytokines, but the mechanisms of cytokine inhibition may vary. A study has reported that minocycline inhibits IκB kinase α/β phosphorylation of NF-κB pathway in THP-1 cells 4 . Another study has reported that clarithromycin attenuates STAT6 phosphorylation 5 . Other studies have reported that macrolide antibiotics inhibited ERK and NF-κB signaling pathways 6,7 . GRNX and MFLX inhibited these signaling pathways to suppress the production of inflammatory cytokines. GRNX significantly inhibited the transcription and secretion of IL-8 induced by LPS-stimulated THP-1 cells by inhibiting ERK1/2 phosphorylation 10 . Furthermore, MFLX inhibited ERK1/2, JNK, and NF-κB activation in the cystic fibrosis epithelial cell line 11 .
Even when using similar quinolone antibacterial drugs, the mechanism of cytokine suppression differs depending on the characteristics of each drug. Previous studies have reported that quinolones with a cyclopropyl group at the N1 position and/or a piperazinyl group at the C7 position, can regulate inflammatory responses [23][24][25] . STFX consists of a fluorocyclopropene at the 1-position of the quinolone skeleton, a chlorine group at the 8-position, a spiroheptane group at the 7-position, and a quinolone with a chlorine group introduced at the 8-position. Such characteristics may cause differences in the spectrum of antibacterial activity and may also cause differences in anti-inflammatory effects.
In the present study, STFX suppressed TNFα production more strongly than the other quinolone antibiotics. It did not suppress the signaling pathways that produced TNFα but increased phosphorylated ERK. Flow cytometry analysis suggested that STFX inhibited the extracellular release of TNFα. TACE specifically cleaves pro-TNFα to release TNFα from cells 26,27 . Our study revealed that STFX reduced the phosphorylation and activity of TACE. One of the mechanisms inhibiting TNFα production by STFX might be interference with TNFα release from cells via the inhibition of TACE activity and phosphorylation but not the inhibition of signaling pathways. www.nature.com/scientificreports/ STFX may be an effective drug for patients with bacterial infections because of its antimicrobial action and the simultaneous reduction of TNFα. STFX has been approved as an oral antibacterial drug and can be used to treat patients with sepsis or septic shock.

Methods
Reagents. Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MFLX, GRNX and CPFX were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). LVFX and STFX were provided by Daiichi Sankyo Company Limited. These antibiotics were diluted with RPMI 1640 at a concentration of 1.0 mg/mL to use as stock solutions. LPS from Pseudomonas aeruginosa serotype 10 (Sigma-Aldrich) was used to induce inflammatory responses. LPS was dissolved in RPMI 1640 medium at a concentration of 1.0 mg/mL and stored at -80 °C until use.
Cell culture and exposures. The human monocyte THP-1 cell line was purchased from the RIKEN Cell Bank (Ibaragi, Japan). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 °C in humidified air with 5% CO 2 and only exponentially growing cells were used for experiments. THP-1 cells (2 × 10 5 cells/mL) were cultured with 0.1 µg/mL of LPS for 4 h, 12 h, 24 h, or 48 h. Data are presented as the mean ± standard deviation (SD) of 6 independent experiments. THP-1 cells (2 × 10 5 cells/mL) were cultured with LPS (0.1 µg/mL) in the presence or absence of antibiotics (MFLX, LVFX, GRNX, CPFX, and STFX) for 4 h. Following the incubation, supernatants were collected via centrifugation at 1500 rpm for 2 min at room temperature and stored at − 80 °C until further analysis. Data are presented as the mean ± SD of 6 independent experiments. ELISA. ELISA was performed using TNFα Human ELISA Kit (Invitrogen, Carlsbad, CA, USA) to determine TNFα concentration. The samples were read using an automated plate reader (Multiskan Spectrum; Thermo Scientific, Waltham MA, USA). Data are expressed as the mean ± SD of 6 independent experiments.